High sensitivity detection of targets and in particular of biomarkers has been a challenge in the field of biological molecule analysis, in particular when aimed at detection of a plurality of targets. Whether for pathological examination or for fundamental biology studies, several methods are commonly used for the detection of various classes of biomaterials and biomolecules.
Some of the techniques most commonly used in the laboratory for detection of single biological targets include gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), western blots, fluorescent in situ hybridization (FISH), Florescent activated cell sorting (FACS), Polymerase chain reaction (PCR), and enzyme linked immunosorbent assay (ELISA). These methods have provided the ability to detect one or more biomarkers in biological samples such as tissues and are also suitable for diagnostic purposes.
However, current global genomic and proteomic analyses of tissues are impacting our molecular-level understanding of many human cancers. Particularly informative are studies that integrate both gene expression and proteomic data. Such multiparameter data sets are beginning to reveal the perturbed regulatory networks which define the onset and progression of cancers (Lin, B.; White, J. T.; Lu, W.; Xie, T.; Utleg, A. G.; Yan, X.; Yi, E. C.; Shannon, P.; Khretbukova, I.; Lange, P. H.; Goodlett, D. R.; Zhou, D.; Vasicek, T. J.; Hood, L. Cancer Res. 2005, 65, 3081-3091. Kwong, K. Y.; Bloom, G. C.; Yang, I.; Boulware, D.; Coppola, D.; Haseman, J.; Chen, E.; McGrath, A.; Makusky, A. J.; Taylor, J.; Steiner, S.; Zhou, J.; Yeatman, T. J.; Quackenbush, J. Genomics 2005, 86, 142-158. Huber, M.; Bahr, I.; Kratzchmar, J. R.; Becker, A.; Muller, E.-C.; Donner, P.; Pohlenz, H.-D.; Schneider, M. R.; Sommer, A. Molec. Cell. Proteomics 2004, 3, 43-55. Tian, Q.; Stepaniants, S. B.; Mao, M.; Weng, L.; Feetham, M. C.; Doyle, M. J.; Yi, E. C.; Dai, H.; Thorsson, V.; Eng, J.; Goodlett, D.; Berger, J. P.; Gunter, B.; Linseley, P. S.; Stoughton, R. B.; Aebersold, R.; Collins, S. J.; Hanlon, W. A.; Hood, L. E. Molec. Cell. Proteomics 2004, 3, 960-969. Chen, G.; Gharib, T. G.; Huang, C.-C.; Taylor, J. M. G.; Misek, D. E.; Kardia, S. L. R.; Giordano, T. J.; Iannettoni, M. D.; Orringer, M. B.; Hanash, S. M.; Beer, D. G. Molec. Cell. Proteomics 2002, 1, 304-313). This new picture of complex diseases such as cancer, and the emergence of promising new cancer drugs (Prados, M.; Chang, S.; Burton, E.; Kapadia, A.; Rabbitt, J.; Page, M.; Federoff, A.; Kelly, S.; Fyfe, G. Proc. Am. Soc. Clin. Oncology 2003, 22, 99. Rich, J. N.; Reardon, D. A.; Peery, T.; Dowell, J. M.; Quinn, J. A.; Penne, K. L.; Wikstrand, C. J.; van Duyn, L. B.; Dancey, J. E.; McLendon, R. E.; Kao, J. C.; Stenzel, T. T.; Rasheed, B. K. A.; Tourt-Uhlig, S. E.; Herndon, J. E.; Vredenburgh, J. J.; Sampson, J. H.; Friedman, A. H.; Bigner, D. D.; Friedman, H. S. J. Clin. Oncology 2004, 22, 133-142.), are placing new demands on clinical pathology (Mellinghoff, I. K.; Wang, M. Y.; Vivanco, I.; Haas-Kogan, D. A.; Zhu, S.; Dia, E. Q.; Lu, K. V.; Yoshimoto, K.; Huang, J. H. Y.; Chute, D. J.; Riggs, B. L.; Horvath, S.; Liau., L. M.; Cavenee, W. K.; Rao, P. N.; Beroukhim, R.; Peck, T. C.; Lee, J. C.; Sellers, W. R.; Stokoe, D.; Prados, M.; Cloughesy, T. F.; Sawyers, C. L.; Mischel, P. S. N. Engl. J. Med. 2006, 353, 2012-2024). For example, traditional pathology practices (i.e. microscopic analysis of tissues) does not distinguish potential responders from non-responders for the new cancer molecular therapeutics (Betensky, R. A.; Louis, D. N.; Cairncross, J. G. J. Clin. Oncology 2002, 20, 2495-2499). Recent examples exist in which pauciparameter molecular measurements are being employed to identify potential responders to at least two therapauetics (Hughes, T.; Branford, S., 2003. Semin Hematol. 2 Suppl 2, 62-68. Lamb, J.; Crawford, E. D.; Peck, D.; Modell, J. W.; Blat, I. C.; Wrobel, M. J.; Lerner, J.; Brunet, J. P.; Subramanian, A.; Ross, K. N.; Reich, M.; Hieronymus, H.; Wei, G.; Armstrong, S. A.; Haggarty, S. J.; Clemons, P. A.; Wei, R.; Carr, S. A.; Lander, E. S.; Golub, T. R., Science 2006, 313, (5795), 1929-1935. Martin, M., Clin. Transl Oncol. 8, (1), 7-14. Radich, J. P.; Dai, H.; Mao, M.; Oehler, V.; Schelter, J.; Druker, B.; Sawyers, C. L.; Shah, N.; Stock, W.; Willman, C. L.; Friend, S.; Linsley, P. S., Proc. Natl. Acad. Sci. 2006, 103, (8), 2794-2799). However, it is unlikely that single-parameter measurements will be the norm. Instead, the coupling of molecular diagnostics with molecular therapeutics will eventually require measurements of a multiparameter (e.g. cells, mRNAs and proteins) biomarker panel that can be used to direct patients to appropriate therapies or combination therapies.
Currently, the measurement of a multiparameter panel of biomarkers from diseased tissues requires combinations of microscopic analysis, microarray data (Mischel, P. S.; Cloughesy, T. F.; Nelson, S. F. Nature Rev. Neuroscience 2004, 5, 782-794), immunohistochemical staining, Western Blots (Mellinghoff, I. K.; Wang, M. Y.; Vivanco, I.; Haas-Kogan, D. A.; Zhu, S.; Dia, E. Q.; Lu, K. V.; Yoshimoto, K.; Huang, J. H. Y.; Chute, D. J.; Riggs, B. L.; Horvath, S.; Liau., L. M.; Cavenee, W. K.; Rao, P. N.; Beroukhim, R.; Peck, T. C.; Lee, J. C.; Sellers, W. R.; Stokoe, D.; Prados, M.; Cloughesy, T. F.; Sawyers, C. L.; Mischel, P. S. N. Engl. J. Med. 2006, 353, 2012-2024), and other methods. The collected data is integrated together within some model for the disease, such as a cancer pathway model (Weinberg, R. A., Cancer Biology. Garland Science: 2006), to generate a diagnosis. Currently, performing these various measurements requires a surgically resected tissue sample. The heterogeneity of such biopsies can lead to significant sampling errors since various measurements of cells, mRNAs, and proteins are each executed from different regions of the tissue.